PE-Operetta介绍.pdf
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PE-Operetta介绍.pdf
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11Operetta高内涵筛选系统刘晓娟HCS产品专员2012-11月22Operetta高内涵成像系统(HighContentImagingSystem)是一个成熟的结合荧光显微成像及多参数定量图像分析的技术。
这种强大的组合为研究人员提供了快速有效的工具,使其能够迅速获得目标蛋白或化合物在细胞中的作用,有效加快了您的决策时间并提高了工作效率。
HCS的概念3320xlongWDobjective,HeLacellsstainedwithAlexaFluor488labeledantitubulinantibodyandDRAQ544Mouseembryonicstemcellcoloniesafter3daysofculturingimagedonOperettaEarlymouseembryonicstemcellcoloniesgrowingonafibroblastfeederlayerimagedonOperetta55Yeast100xObjective66Brightfield#2c.elegansbrightfield77eGFP#2c.elegansheatshockinducedGFP88ZebrafishBrightfieldGFPVascularStructuredsREDRedBloodCells99Immunohistochemistry(IHC)1010SmallOrganismImaging1111WholeOrganismImagingZebrafishembryo,4dayspostfertilizationC.elegansDICGiveshigherresolutionandopticalsections,Butgenerallynotusefulforplasticsubstrates1212专门为HCS设计的光路高QE14bitCCD适应HCS定量需求自动化物镜与滤光片管理系统300W高能氙灯740nmLED明场光源三种成像光路远红外高速自动对焦长工作距离和高数值孔径物镜可供选择适应各种不同微孔板的成像要求XenonlampAttenuatorExcitationfilterwheelEmissionfilterwheelDichroicmirrorwheelCameraTransmissionlightsourceAutofocusunitOptionalconfocallightpath,pinholediscShutterShutterObjectiveturretSampleOperetta硬件配置1313高能量300W,连续波长可用波段内无能量突变自适应光路,无需校正维护方便简单氙灯光源otherlampsOperettalamp1414八个激发光滤光片转轮,(360640nm)八个条形码自动识别的发射光滤光片位置15个不同波长的滤光片可供选择维护窗口全光谱覆盖的光路15152X-100X物镜可供选择有Pre-scan功能,方便用户扫描特定孔板区域条形码自动识别全自动物镜工作距离判定与保护,防止物镜受损高数值孔径物镜符合高质量图像要求长工作物镜适应各种不同规格的微孔板Objectivechoices:
2XNA0.08,WD12mm10xNA0.3,WD10mm10xNA0.4,WD3.1mm20xNA0.45,WD6.67.8mm20xNA0.75,WD0.6mm40xNA0.6,40xNA0.9560xNA0.7,60xNA0.90100xNA0.85齐全的物镜1616特别设计的光路使用户可以更高效率的捕获细胞,而不损失分辨率有效减少用户需要拍摄的照片数量Typicalfieldofviewusing20xobjectiveLargefieldofviewwiththeOperettausing20xobjectiveOperettafieldofview10x:
1.04x1.29mm20x:
518x645m40x:
260x323m60x:
173x215m超大视野,提供更高细胞捕获效率1717Hela的共聚焦图像CollaborationwithUlrichRothbauerNuclearlamina,visualizedbyaGFP-taggedChromobodyinHeLacellsWidefieldfluorescenceConfocalfluorescence1818Mitochondria60xhighNAOperettaNon-confocalconfocalBackground:
4008001919Confocal-pinholediscsuppressesoutoffocusfluorescenceCellularstructuresinfocalplanebecomemorequantitative,clearandcrispDrosophilaEmbryo20xLWDWidefieldConfocalConfocalFluorescence2020HCS的多参数分析2121Acellcantelluswhat?
FluorescenceQuantitationMorphologyCaculationTextureAnalysis2222CellMorphology:
AreaWidthLengthRoundnessWidth/LengthratioIntensity:
MeanIntensityIntensityVariation(stddev,CV%)RadialIntensityDistributionTexture:
SER8featuresGabor2featuresHaralick4featuresQuantificationofSpotoccuranceFibreoccuranceFibrealignmentPatchoccuranceSmooth/irregulardistributionPatternoccuranceDegreeofgranularityAvailableParametersforCellularFunctionOuruniquesetofmorphometric,intensity-basedandtextureparametersprovidesacomprehensivesolutionforquantifyinge.g.cytoskeletalrearrangementandspindleformation2323Apictureisworthathousandwords!
membranefluorescenceligandbindingapoptosiswholecellfluorescencetargetphosphorylationtransporteractivitycytotoxicitymembranepotentialnuclearmorphology/stainingapoptosismitoticindex/stageotherscustomizedassaysbead-basedmRNAquantificationandcombinationscytosol-nucleustranslocationproteinkinaseactivationtranscriptionfactoractivationcellmorphologyneuriteoutgrowthapoptosiscytotoxmembranecytosoltranslocationreceptorinternalizationrecruitmentofsignalingproteins2424HCS的优点每个细胞都可以筛选是否可以使用于分析,比如定义细胞大小、形状及荧光强度对细胞进行筛选。
-消除了背景,如颗粒及细胞碎片所带来的背景值。
应用范围广2525HCS的技术优势HCS与流式:
流式将细胞看作一个质点,只能检测标记的荧光强度,不能将细胞进行分区域分析HCS与ELISA:
HCS可以清楚看到细胞内所发生的所有反应,结果更加直观,更加可靠HCS与Reportergene:
HCS获得的多参数结果是Reportergene实验不能实现的,它可以在亚细胞结构上研究蛋白与蛋白之间的相互作用,背景更低HCS与牛痘病毒中和实验传统的牛痘病毒中和实验,要等待48-72个小时进行空斑形成,而且需要靠人进行空斑的计数。
HCS利用带有GFP基因的病毒感染细胞,不需要空斑形成这些复杂的步骤,计算病毒滴度更方方便,更准确2626HCS在传染病领域的应用病毒转染效率的检测:
DAPI染核,病毒转染系统用GFP融合蛋白。
病毒滴度的检测:
带有GFP的病毒滴度的检测,可以不用plaque,节省时间可以用瞬时转染系统取代稳定筛选的系统进行研究:
稳定筛选的细胞系由于过表达可能对细胞产生毒性,而且HCS使瞬时转染摆脱了转染效率的影响RNAi文库的筛选:
对病毒感染机制进行研究抗病毒药物的筛选:
2727HarmonySoftware2828PackagedReadyMadeSolutionsforoneclickAnalysis2929Selectbuildingblockfromdrop-downmenuanalysisisimmediatelyappliedonimageClickonacelltoseecorrespondingdatainthetableBuildingBlock:
FindNuclei3030ChoosefromasetofmethodsProgressivedisclosureofoptionaltuningparametersAutomaticscanforparametersettings,instantfeedbackontheimageAdjustParameters3131Selectbuildingblockfromdrop-downmenuanalysisisimmediatelyappliedonimageFindCytoplasm3232HarmonyautomaticallyprovidesbestfitparametersettingManualadjustmentispossibleFinds“Best”ParameterAutomatically3333SelectCellRegion3434TextureforImageAnalysissmoothgranularLookingfordifferentintensityvaluesinneighboringpixelspatchesExamples:
ZernikeMoments:
Statisticsinasingleobject(e.g.standarddeviation)ThresholdAdjacencyStatistics(TAS):
IntensityrelationsofneighboringpixelsHaralick:
Intensityrelationsoverlargerpixelarrays.3535ActinstainingDetectedfibresDetecteddegreeoffibrealignement00.0050.010.0150.02Cell68Cell55ScoreValue00.010.020.030.040.050.06Cell68Cell55ScoreValueOccuranceoffibresAlignmentoffibresAnalysisofCytoskeletalRearrangementSERRidge“parameterenablesquantificationoffibreoccurance(left).Atexturefilter-feature“GaborMax”-respondstofibrealignment(right).3636ConventionalAnalysis:
BasedonPlasmaMembraneRegionConventional-plasmamembranestainbasedevaluationofcytosoltomembranetranslocationisamajorchallengeformostimageanalysissoftware.Membraneregionsegmentation,basedonplasmamembranestain,isdifficult3737HarmonyAnalysis:
BasedonTextureParameterSERRidge“Ouruniquetextureparameterbasedimageanalysisapproachcandetect“Ridge”structuresofplasmamembranereproducibly3838SpleenTissueSectionmountedon1x3”SlidewithCoverslip10xMagnification8ImageMontageBlueDAPIYellowPhalloidinSharedwithpermissionfromthelaboratoryofDr.PatrickKaminkerHumanGenomeSciencesInstituteRegionofInterest393920xMagnificationBrightfield404020xMagnificationDAPI414120xMagnificationPhalloidin4242434320xMagnification3ColorOverlay4444AllCellsSegmented4545CellsSelectedbasedonIntensityOnly4646Notethedifferencein“texture”ofthecellcircledinRedvsWhite4747CellsSelectedbasedonIntensityandTextureAnalysis4848MicronucleusDetectionVariousSizedSpotsWhattodo4949划时代的智能化软件人工智能人工智能机机器自主学器自主学习习远远离离HCS复复杂参数杂参数初学者初学者/研究生快速掌握研究生快速掌握5050MACHINELEARNING(PhenoLOGIC)thatWORKS!
PhenoLOGICUseMachineLearningto:
Analyzebrightfieldimages(labelfreedetection/analysis)TeachsoftwaretodifferentiatepopulationintonumeroussubpopulationsbysimplyclickingoncellsorareasofanimageAutomaticallyincorporatesallattributes,includingvarioustexturefeaturesNoneedtotryvariouscalculationsoroptimizetheinputsettings:
allcalculationsandsettingoptimizationsareperformedforyoubyPhenoLOGIC!
5151OrisCellMigrationAssayAssayOris96-wellCollagenIcoatedplateswithOrisCellSeedingStoppers;twodifferentcelllines:
HT-1080MDA-MB-231Incubationfor6h;removestoppers;Migrationfor18h;fixationStainingwithDAPIandTRITC-phalloidinMeasurementQuantifyopenarearemaningMDA-MB-231pre-migrationMDA-MB-231post-migration5252TheFuture:
Label-FreeAnalysisofProliferationOperettabrightfieldimageCalculatedtexturefeature“granularity”fortheimageSegmentedimage:
covered(green);free(yellow)Teachingtextureclass“A”(yellowcircles)andtextureclass“B”(greencircles)5353KeytechnologyMachineLearningforeasytouse!
54545555PhenoLOGICidentifiesapairofparameterstodistinguishbetweenlivinganddeadcellsClassificationofcellviabilitybythePhenoLOGIClinearclassifiercanreplacetheBOBO-3deadcellstainIdentificationofredundantstainingsavestimeandreducescostsPhenoLOGIClinearclassifierreplacesadditionalstainingLive/deadcellclassificationusingPhenoLOGICMitochondrialtextureSERRidge2px(arbitraryunits)Cellaream-0.0050.0150.0050.02501000500150020002500FCCPM%Deadcells-3-2-10123020406080100PhenoLOGIC(EC501.13M)BOBO-3(EC501.34M)5656PowerfulneuriteanalysisLeft:
individualsegments(differentcolors)Right:
Roots(circles),nodes(arrows),andends(numbers)UniquecolocalizationanalysisNeuritescanbeusedassearchmaskforsignalscolocalizedonneuritesessentialforaxonanalysisMiddle:
maskcreatedfromneuritesLower:
Spotidentificationonneurite(arrow)神经分析软件5757STARMorphologypropertiesAmorecomplete,statisticallyrobustsetofpropertiesforcomprehensivelydescribingcellmorphology,distributionofintensitywithinregions,.Forhigherqualityassays.5858STAR=Symmetry,ThresholdCompactness,Axial,RadialBuildingBlock:
CalculateMorphology-STARSymmetryThresholdCompactnessAxialRadialProfile5959ExamplestoillustratetheZrangeExamplescreenshotsfromascreenanalyzedwithColumbusbadZacceptableZ6060Example-TransfluorGPCRinternalizationUntreatedActivatedCelltype1ActivatedCelltype2ScreenshotofColumbusAssayDefinition6161ComparisonofdifferentImageAnalysisAlgorithms.isverysimpleusingtheCalculateZprime“buildingblockHandtailoredimageanalysisbasedonspotdetectionBestanalysisautomaticallyidentifiedbyColumbususingtheSTARmorphologyfeaturesetandSecondaryAnalysistochoosethebestfeatureZ=0.60Z=0.666262Assessmentofanalysisrobustnessfordifferentcelltypes.isverysimpleusingVariations“inSecondaryAnalysisCelltype1Celltype2Onlyonemouseclickne
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